bioRxiv preprint first posted online Jun. 17, 2015; doi: http://dx.doi.org/10.1101/020958. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Full title: Evolution and coexistence in response to a key innovation in a long-term evolution
experiment with Escherichia coli
Running title: Coexistence in response to a key innovation
Key words: adaptation, experimental evolution, cross-feeding, evolutionary tinkering, citrate, C4dicarboxylates
Authors:
Caroline B. Turner
Ecology, Evolutionary Biology and Behavior Program, Michigan State University, East Lansing,
MI
Zachary D. Blount
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing,
MI
Daniel H. Mitchell
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing,
MI
Richard E. Lenski
BEACON Center for the Study of Evolution in Action; Department of Microbiology and
Molecular Genetics; and Ecology, Evolutionary Biology and Behavior Program; Michigan State
University, East Lansing, MI
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bioRxiv preprint first posted online Jun. 17, 2015; doi: http://dx.doi.org/10.1101/020958. The copyright holder for this preprint (which was not
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Abstract
Evolution of a novel function can greatly alter the effects of an organism on its environment.
These environmental changes can, in turn, affect the further evolution of that organism and any
coexisting organisms. We examine these effects and feedbacks following evolution of a novel
function in the long-term evolution experiment (LTEE) with Escherichia coli. A characteristic
feature of E. coli is its inability to consume citrate aerobically. However, that ability evolved in
one of the LTEE populations. In this population, citrate-utilizing bacteria (Cit+) coexisted stably
with another clade of bacteria that lacked the capacity to utilize citrate (Cit–). This coexistence
was shaped by the evolution of a cross-feeding relationship in which Cit+ cells released the
dicarboxylic acids succinate, fumarate, and malate into the medium, and Cit– cells evolved
improved growth on these carbon sources, as did the Cit+ cells. Thus, the evolution of citrate
consumption led to a flask-based ecosystem that went from a single limiting resource, glucose, to
one with five resources either shared or partitioned between two coexisting clades. Our findings
show how evolutionary novelties can change environmental conditions, thereby facilitating
diversity and altering both the structure of an ecosystem and the evolutionary trajectories of
coexisting organisms.
2
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Evolution does not produce novelties from scratch. It works on what already exists, either
transforming a system to give it new functions or combining several systems to produce a more
elaborate one.
–Francois Jacob
Introduction
Although ecology and evolutionary biology are often treated as distinct disciplines, scientists as
far back as Darwin have recognized that the two are closely intertwined (Darwin 1881; Dawkins
1982; Lewontin 2001). Recent research on the interplay between ecological and evolutionary
processes (Matthews et al. 2011; Schoener 2011) has emphasized that the two can interact even
over short time scales. Evolutionary innovations—and in particular the emergence of
qualitatively new functions—have the potential to alter ecological conditions both for the
organisms that evolved the innovation and for coexisting organisms. Perhaps the most dramatic
example of this interplay in the history of life on Earth was the evolution of oxygenic
photosynthesis. This new way of obtaining energy from light was extraordinarily successful, but
it produced oxygen as a by-product. Over time, oxygen accumulated in the atmosphere, where it
acted as a toxin to most extant life (Knoll 2003; Sessions et al. 2009). Many organisms were
likely driven extinct, but others evolved mechanisms to tolerate and use oxygen; thus, the
oxygenated world created new ecological niches requiring aerobic respiration and perhaps
favoring multicellularity (Schirrmeister et al. 2013).
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The origin of a novel function is, by its nature, a rare event. Examples such as the
evolution of oxygenic photosynthesis can only be studied using the fossil and geological records,
making it difficult to resolve the order of events and examine the consequences of the
innovation. The evolution of a new function can lead to major changes in an ecosystem, as some
lineages may be driven extinct while others evolve into new niches. Ideally, one would like to be
able to study the ecological and evolutionary consequences of an evolutionary innovation by
directly comparing the properties of living organisms from before and after the innovation and
measuring their relative fitness by competing the ancestral and derived forms in the
environments where they evolved. In fact, the evolution of a novel function in a laboratory
evolution experiment gives us the opportunity to do just that.
The long-term evolution experiment with Escherichia coli (LTEE), in which this novel
function arose, is an ongoing experiment in which 12 populations founded from a common
ancestor have been evolving in identical environments for more than 25 years. The initial
ecology of the LTEE was deliberately made very simple, with the bacteria growing on a single
carbon source, glucose, in a carbon-limited medium (Lenski et al. 1991). The culture medium
also contained a large quantity of a potential second carbon source, citrate, but this resource was
not available for E. coli metabolism. In fact, the inability to grow aerobically on citrate is one of
the diagnostic features of E. coli (Koser et al. 1924). However, after more than 30,000
generations, one of the populations, called Ara-3, evolved the ability to use the citrate (Blount et
al. 2008). After 33,000 generations, the citrate-consuming bacteria (Cit+) became numerically
dominant in the population, and the total population size increased several fold. Following this
large increase in numbers, much of the existing diversity in the population was eliminated, but a
clade that could not consume citrate (Cit–) persisted and exhibited negative frequency-dependent
4
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coexistence with the Cit+ cells (Blount et al. 2008; Blount et al. 2012). The clade of Cit– cells that
persisted and the clade in which the Cit+ phenotype arose had diverged about 10,000 generations
before the origin of the Cit+ function. All of the Cit– cells isolated after the Cit+ type became
numerically dominant came from this same clade (Blount et al. 2012).
We set out to study the ecological consequences of the evolution of citrate consumption
and how it affected the further evolution of both the Cit+ and Cit– lineages. In particular, we
focused on studying the mechanism by which the Cit– clade coexisted with the Cit+ clade. At
least two distinct mechanisms could underlie this coexistence. First, a tradeoff between growth
on glucose and citrate might allow the Cit– lineage to persist as a specialist on glucose. Indeed, a
comparison of the growth curves of Cit+ and Cit– cells showed that the Cit+ cells had a longer lag
time when growing under the same conditions as used in the LTEE (Blount et al. 2008). Second,
Cit– cells might cross-feed on one or more carbon compounds released by the Cit+ cells. We
define cross-feeding as the consumption by one group of organisms of products produced by
another group of organisms. The two mechanisms are not mutually exclusive and could act
together to enable the coexistence of the Cit+ and Cit– lineages.
The emergence of cross-feeding interactions has been observed repeatedly in evolution
experiments with E. coli (Rosenzweig et al. 1994; Treves et al. 1998), and it is predicted to occur
by theory under certain conditions (Doebeli 2002). Cross-feeding occurs when, in the process of
growing on a primary resource, one bacterial ecotype secretes a byproduct that is consumed by
another ecotype. Cross-feeding is favored when bacteria can grow more quickly by not fully
degrading their primary resource, but instead secrete incompletely digested byproducts, opening
the opportunity for the evolution of specialists on the byproducts. Cross-feeding has evolved in
at least one other population of the LTEE, in which two lineages have stably coexisted for tens of
5
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thousands of generations (Rozen and Lenski 2000; Le Gac et al. 2012); in another population, a
negative frequency-dependent interaction arose, probably also involving cross-feeding, that led
to transient coexistence for several thousand generations (Maddamsetti et al. 2015; Ribeck and
Lenski 2015). However, cross-feeding interactions may be less common in the LTEE than in
some other evolution experiments because the relatively low glucose input and resulting low
bacterial density limit the accumulation of secondary metabolites (Rozen and Lenski 2000).
Moreover, mathematical modeling shows that cross-feeding will be less favored under a serialtransfer regime like that of the LTEE, where population densities are low much of the time, than
in continuous culture, where populations are always near their maximum density (Doebeli 2002).
One potentially important set of molecules for cross-feeding in the Cit+/Cit– system
includes the C4-dicarboxylic acids succinate, fumarate, and malate. The Cit+ phenotype is based
on the aerobic expression of the gene encoding the CitT transporter protein (Blount et al. 2012).
CitT is a generalized di- and tricarboxylic acid antiporter that can import citrate, succinate,
fumarate, and malate in exchange for export of any of these molecules (Pos et al. 1998). Thus,
any net import of citrate into the cell requires that the CitT transporter pump succinate, fumarate,
or malate out of the cell, making these C4-dicarboxylates likely molecules for cross-feeding.
Here we show that a key innovation in a long-term experiment with E. coli, the evolution
of aerobic citrate metabolism, altered the ecological conditions experienced by the bacteria,
influencing the subsequent evolution of both the lineage that evolved the new function as well as
another surviving lineage that did not. In particular, we demonstrate that Cit+ cells modified the
environment by releasing additional carbon sources—the C4-dicarboxylates—into the medium.
The coexisting Cit– bacteria evolved to cross-feed on those resources, adapting to the modified
environment but resulting in a cost to their fitness when growing on glucose in the absence of the
6
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Cit+ cells. The Cit+ bacteria also evolved improved growth on the additional resources, thus
competing with the Cit– lineage for both the glucose and C4-dicarboxylates.
Methods
Long-term evolution experiment with E. coli
The long-term evolution experiment (LTEE) consists of 12 evolving populations of E. coli B,
which are serially propagated in 10 mL of Davis minimal medium with 25 mg/L glucose
(DM25). All populations were initiated from two founder clones that differ by a neutral
mutation. Cultures are grown in a 37°C shaking incubator under well-mixed, aerobic conditions.
Growth in DM25 is carbon-limited with a final population density of ~5 x 107 cells/mL in the
absence of citrate consumption. Growth of Cit+ cells remains carbon-limited (Fig. S1) with a
population density of ~2 x 108 cells/mL. Each population is diluted 1:100 into fresh medium
daily, allowing regrowth of the population for ~6.6 generations per day. Every 500 generations,
samples of each population are frozen at -80°C. These frozen organisms are viable and can be
revived for later experimentation. More details on the methods used in the LTEE are given in
Lenski et al. (1991).
Strains used and conditioning procedure
Other than the ancestral clone, REL606, all clones were isolated from the LTEE population,
designated Ara-3, in which the ability to consume citrate evolved. Unless otherwise specified,
the Cit– clones used in this study were from the Cit– clade (called clade 2 in Blount et al. 2012)
that persisted after the evolution of citrate consumption. Clones from the Cit+ clade vary in their
7
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ability to grow on citrate. All clones from the Cit+ clade (called clade 3 in Blount et al. 2012)
used in this study that were isolated at 32,000 generations or later have a Cit+ phenotype;
however, clones from that clade that were isolated at earlier time points have a Cit– phenotype.
Members of the Cit– and Cit+ clades were identified by mutations unique to each lineage, as
described in Blount et al. (2012). Table S1 lists all of the strains used in this study.
Prior to initiating each experiment, we revived samples of frozen isolates by first growing
them in Luria-Bertani broth (LB). We then conditioned them to the environment of the LTEE by
growing them for 2 days in DM25, with transfers into fresh medium every 24 h.
Resource assays
We hypothesized that the Cit+ cells released C4-dicarboylates into the culture medium, as a
consequence of the antiporter mode of action of the CitT protein. To test this hypothesis, we
employed gas chromatography–mass spectrometry (GCMS) to measure the concentrations of
succinate, fumarate, and malate in the medium over the course of a 24-h transfer cycle. We
measured these C4-dicarboxylate concentrations in cultures of the following strains: REL606, the
founding strain of the Ara-3 population; a 40,000-generation clone from the Cit– clade; and eight
clones from the Cit+ clade sampled every 2,000 generations from 30,000 to 44,000 generations.
Of the clones from the Cit+ clade, the 30,000-generation clone had a Cit– phenotype, the 32,000generation clone exhibited weak growth on citrate, and the remaining clones exhibited strong
growth on citrate. After reviving and conditioning the clones as described above, we transferred
the cultures to fresh DM25 medium. At 0, 2, 4, 6, 8, 12 and 24 h after transfer, 300 mL of each
culture was collected using a sterile syringe, immediately filter-sterilized by passing the culture
medium through a 0.25-µm filter, and frozen at -80°C for later analysis.
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We measured the succinate, fumarate, and malate concentrations in these samples using
an Agilent 5975 GCMS system at the Michigan State University Research Technology Support
Facility. Because DM25 medium contains a high concentration of phosphate that obscured all
other molecules in full-scan mode, we could not conduct a broad chemical survey. We instead
used selective ion measurement to isolate the spectral signatures specific to succinate, fumarate,
and malate. Because of this limitation, we cannot eliminate the possibility that Cit+ cells also
released other molecules into the culture medium.
We also measured the concentration of citrate in the medium to compare the timing of
citrate consumption to the timing of succinate, fumarate, and malate release. We measured citrate
consumption by the 30,000-, 32,000-, 34,000- and 40,000-generation Cit+ clones with five-fold
replication. We collected filtered samples of culture medium at 0, 1, 2, 3, 4, 6, 9, and 24 h,
following the same protocol as above. We then measured the concentration of citrate in these
samples using a Megazyme citric acid assay kit.
Growth using C4-dicarboxylates as carbon sources
To assess changes in the ability of clones from the Cit– and Cit+ clades to grow on succinate,
fumarate, and malate, we analyzed growth curves for Cit– and Cit+ clones isolated every 1,000
generations from 30,000 to 43,000 generations. This range includes clones from three periods of
interest: (i) prior to the origin of the Cit+ phenotype (30,000 to 31,000 generations; (ii) when Cit+
cells were present, but very rare (32,000 to 33,000 generations); and (iii) after the several-fold
population expansion of the Cit+ lineage (34,000 to 43,000 generations). The growth trajectories
of each clone were assessed for three replicate cultures in media with a single alternative carbon
source in the place of glucose. In addition to the C4-dicarboxylates succinate, fumarate, and
9
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malate, we also measured the growth of the Cit– clones on acetate, a common substrate for crossfeeding in E. coli (Rosenzweig et al. 1994; Treves et al. 1998).
We used glucose as a carbon source during conditioning so that all cultures would have a
similar initial density. For the Cit– clones, growth curves were conducted in DM medium.
However, growth curves of the Cit+ clones were conducted in M9 minimal salts medium
(Sambrook and Russell 2001) because DM medium contains citrate, which would confound the
assays. As a result, the growth curves for the Cit+ and Cit– clones are not directly comparable;
instead, comparisons of the growth curves provide information on the changes that evolved
within each clade over time. The concentrations of the carbon sources used were as follows: 30.5
mg-succinate/L, 39.5 mg-fumarate/L, 45.7 mg-malate/L, and 34.4 mg-sodium acetate/L. We
chose these concentrations because they give stationary-phase population densities similar to
those in DM25. Growth curves were generated in 96-well microplates by measuring optical
density (OD) at 420 nm every 10 min for 24 h (or 48 h, where noted) using a VersaMax
automated plate reader (Molecular Devices).
We analyzed two characteristics of the growth curves: the final OD value at 24 h, and the
time required to reach stationary phase. We determined time to stationary phase by visually
inspecting each log-transformed growth curve and identifying the time point when a population
switched from exponential growth to either a stable or declining OD. These visual assessments
were performed twice, in a blind fashion, and the results were extremely consistent between
repetitions.
Fitness assays
10
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To determine how evolutionary changes in the Cit– lineage affected the fitness of Cit– cells in the
presence and absence of Cit+ cells, we conducted a series of competition experiments between a
reference 30,000-generation Cit– clone and Cit– clones sampled every 1,000 generations from
30,000 to 40,000 generations. All Cit– clones used in these assays were from the Cit– clade. All
of the Cit– clones competed against the reference clone, in both the presence and absence of a
mutant derived from a Cit+ clone from 40,000 generations, with three replicates of each
competition. Competitions ran for one standard 24-h cycle, and fitness values were calculated as
the ratio of realized population growth rates during the competition assay (Lenski et al. 1991).
The Cit– clones used were the same as those for the growth measurements above, with the
exception of 33,000-generations, for which we inadvertently used a different clone, CZB195,
instead of CZB194. However, we confirmed in separate measurements that CZB195, like
CZB194 was unable to grow on the C4-dicarboxylates.
The reference clone, called CBT1, was an arabinose-consuming (Ara+) mutant of a Cit–
clone isolated from the 30,000-generation population (Table 1). Arabinose consumption is a
neutral marker under the conditions of the LTEE (Lenski et al. 1991) that allows the
differentiation of bacteria by their colony color when grown on tetrazolium arabinose (TA) agar
plates (Levin et al. 1977; Lenski et al. 1991). To isolate CBT1, we grew the 30,000-generation
Cit– clone in 10 mL of DM25 medium, then centrifuged the culture and plated the concentrated
cells onto a minimal arabinose agar plate. After incubation, a single colony from the plate was
streaked twice on minimal arabinose agar plates. A colony from the second plate was grown in
LB, and a sample of that culture was frozen. We then confirmed that CBT1 was selectively
neutral compared to its parent clone by conducting a seven-day competition between the two
clones under the same conditions as the LTEE.
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To conduct competitions between Cit– clones in the presence of Cit+ cells, we needed a
way to prevent the Cit+ bacteria from growing on the TA plates. To this end, the competitions
were conducted using a λ-sensitive mutant, called CBT3, of a Cit+ clone isolated from the
population at 40,000 generations. Although the founding clone of the LTEE was sensitive to
bacteriophage λ, resistant mutants were favored and went to fixation in population Ara-3 by
10,000 generations (Meyer et al. 2010). Resistant strains exhibit reduced expression of LamB, a
maltose transport protein that is also the adsorption site for λ (Pelosi et al. 2006; Meyer et al.
2010). To isolate the λ-sensitive mutant, CBT3, we selected for a maltose-consuming mutant
following the procedure described above for isolating an Ara+ mutant, substituting minimal
maltose for minimal arabinose agar plates. We confirmed that CBT3 was sensitive to phage λ by
plating onto TA plates with and without λ. The fitness of the Cit+ λ-sensitive mutant was similar
to that of its λ-resistant parent in one-day competition assays (relative fitness 0.98 ± 0.06, mean ±
95% confidence interval, n = 10), and the Cit+ population size was unchanged.
For all competitions between Cit– clones that were performed in the presence of Cit+
cells, the cultures were plated together with phage λ to prevent the growth of Cit+ colonies.
However, a few Cit+ colonies were able to grow on these plates, presumably due to mutations to
λ-resistance or incomplete coverage of λ on the plates. To correct for this problem, we
transferred a sample of all Ara– colonies onto Christensen’s citrate agar, an indicator medium for
the Cit+ phenotype (Reddy et al. 2007). Any colonies that showed a positive reaction on
Christensen’s agar were excluded from the counts used to measure the relative fitness of the Cit–
competitors.
Based on the outcome of these experiments, we decided to compete Cit– clones isolated
at 33,000 and 34,000 generations directly against one another. We measured the relative fitness
12
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of the 33,000- and 34,000-generation clones with ten-fold replication in both the presence and
absence of Cit+, following the same procedure as above. Half of the replicates had the 33,000generation clone competing against an Ara+ mutant of the 34,000-generation clone (named
CBT2, isolated as described above), and half had the 34,000-generation clone competing against
an Ara+ mutant of the 33,000-generation clone (called CZB207).
Genomic analysis
The genomes of seven clones from the Cit– clade were sequenced by Blount et al. (2012). We
inspected the genome sequences to look for candidate mutations that might have affected the
evolution of C4-dicarboxylate consumption. Three clones—ZDB357, ZDB200, and ZDB158
from generations 30,000 to 32,500 (Table S1)—could not grow on the C4-dicarboxylates. The
other four clones—ZDB87, ZDB99, ZDB111, and REL10988 from generations 36,000 to 40,000
(Table S1)—exhibited strong growth on the C4-dicarboxylates. We used the breseq 0.23 software
(Deatherage and Barrick 2014) to generate a list of mutations that were present in the C4dicarboxylate-consuming clones, but absent in the clones that lacked the ability to grow on C4dicarboxylates.
Genetic manipulation
We constructed an isogenic derivative of the ancestral clone REL606 that carried the dcuS allele
found in a 34,000-generation Cit– clone called ZDB86. To do so, we used the pKOV plasmid and
the methods in Link et al. (Link et al. 1997). We performed Sanger sequencing to confirm that
the evolved dcuS allele was present in the resulting construct, ZDB1052.
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Results
Resource assays
Consistent with our expectation based on the antiporter mechanism used for citrate uptake, we
found that Cit+ clones secrete succinate, fumarate, and malate into the culture medium. The
concentrations of these C4-dicarboxylates remained low throughout the 24-h transfer cycle in
cultures of the ancestral strain (REL606), the 40,000-generation Cit– clone, and a 30,000generation clone from the clade that later gave rise to the Cit+ lineage (Fig. 1). By contrast, both
succinate and fumarate concentrations were elevated in all cultures of all of the Cit+ clones
during at least part of the 24-h cycle (Fig. 1A-B). Malate concentrations were also noticeably
elevated in all except one (from generation 36,000) of the Cit+ clones that we analyzed (Fig. 1C).
The peak succinate levels in medium with Cit+ clones from 34,000 generations and later
occurred at 4 or 6 h after inoculation and ranged from 0.9 to 2.1 mg/L (Fig. 1A). Succinate
concentrations then declined to below detectable levels by 8 h. The culture medium of the
32,000-generation Cit+ clone, which grows weakly on citrate, showed a very different pattern,
with a peak succinate concentration of 2.1 mg/L at 12 h and 1.3 mg/L remaining at 24 h.
Fumarate and malate exhibited similar patterns to succinate, with peak fumarate
concentrations ranging from 0.6 to 1.1 mg/L and peak malate concentrations from 3.2 to 11.0
mg/L (Fig. 1B-C). (The 36,000-generation Cit+ clone was atypical, however, in that it did not
produce any measurable amount of malate.) As with succinate, the 32,000-generation, weakly
Cit+ clone released fumarate and malate, but did not fully consume them from the medium. None
of the C4-dicarboxylates showed any obvious trend, upward or downward, in peak concentration
over evolutionary time.
14
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Fig. 2 shows the concentration of citrate in the culture medium during the growth of
several Cit+ clones. The earliest Cit+ clone from generation 32,000 drew down citrate only
slightly, if at all, over the full 24-h transfer cycle. By contrast, both Cit+ clones that we tested
from generations 34,000 and 40,000 rapidly removed citrate in the period from 4 to 6 h, which
coincides with the period when the C4-dicarboxylates reached their highest levels (Fig. 1). By 9
h, both these clones drew down the citrate concentration to below the detection limit.
Growth on C4-dicarboxylates
Clones from the Cit– clade isolated before the demographic shift (30,000–33,000 generations)
did not exhibit a measurable increase in OD even after 24 h in glucose-free medium
supplemented with any of the three C4-dicarboxylates (Fig. 3A-C). However, all Cit– clones from
34,000 generations and later showed improved growth on succinate, fumarate, and malate with
OD values significantly greater than zero after 24 h (Fig. 3D-F). These later clones also reached
stationary phase in fewer than 24 h, and the time they took to reach stationary phase declined
between 34,000 and 43,000 generations (Fig. 3D-F).
Clones from the Cit+ clade also showed negligible growth on C4-dicarboxylates through
32,000 generations (Fig. 4A-C). Beginning at 33,000 generations, Cit+ clones had significantly
improved growth on succinate, fumarate, and malate as sole carbon sources (Fig. 4A-C). The
32,000-generation clone had a weak Cit+ phenotype, but it showed no growth on succinate,
fumarate, or malate, indicating that the ability to grow on these carbon sources was not simply a
pleiotropic effect of the same mutations that allowed growth on citrate. Unlike the clones from
the Cit– clade, Cit+ clones did not show consistent improvement across the generations in their
growth in succinate medium. For example, the Cit+ clones from generations 35,000, 38,000 and
15
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46,000 had lower final OD values than other late-generation Cit+ clones and did not reach
stationary phase within 24 h when growing on succinate (Fig. 4D). Growth of the Cit+ clones on
fumarate and malate was similarly variable (Fig. 4E-F), but the particular clones with slower
growth or lower final density differed across the three substrates.
In contrast to the pattern of improved growth on the C4-dicarboxylates succinate,
fumarate, and malate after the demographic expansion of the Cit+ lineage, clones from the Cit–
clade showed reduced growth on acetate, both in terms of final OD values (Fig. 5A) and time to
stationary phase (Fig. 5B). Thus, acetate cross-feeding is unlikely to contribute to the coexistence
of the Cit– and Cit+ lineages. Moreover, the reduced growth on acetate indicates that the
improved growth of Cit– clones on C4-dicarboxylates is specific to those molecules, rather than a
general improvement in either growth or the ability of Cit– cells to shift from growing on glucose
to other carbon resources.
Fitness assays
If the Cit– lineage has evolved to feed on byproducts secreted by the Cit+ lineage, then it should
show evidence of evolving higher fitness, relative to its Cit– predecessors, in the presence of Cit+
cells than in their absence. We tested this prediction by competing clones from the Cit– clade
isolated at generations 30,000 and 32,000, and then every 1,000 generations from 33,000 to
43,000 generations, against a reference Cit– clone from 30,000 generations, both in the presence
and absence of a common Cit+ clone that could be excluded when counting the two Cit–
competitors (see Methods). In the presence of Cit+ cells, the fitness of the Cit– clones trended
higher in later generations, with mean fitness values relative to the 30,000-generation reference
significantly greater than unity for 9 of the 11 time points tested from generations 33,000 to
16
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43,000 (Fig. 7). However, in the absence of Cit+ cells, the fitness of the Cit– clones actually
declined over time (Fig. 6). These results not only support the cross-feeding hypothesis, but they
also suggest that the adaptation of the Cit– lineage to the cross-feeding niche imposed a tradeoff
with respect to growth in its previous glucose-only niche.
Although the overall trend in these data is consistent with our expectations, it is
surprising that the increase in the fitness of the Cit– clones, when assayed in the presence of Cit+
cells, seems to have begun by 33,000 generations (Fig. 6), whereas we did not see any evidence
of their growth on C4-dicarboxylates until generation 34,000 and later (Fig. 3). If the differences
in fitness of the Cit– cells in the presence and absence of Cit+ cells were caused by the evolution
of the capacity for growth on the C4-dicarboxylates, then we would expect the 34,000-generation
C4-dicarboxylate-consuming Cit– clone to be more fit than the 33,000-generation Cit– clone in
the presence of Cit+ cells and, perhaps, less fit in the absence of Cit+ cells. This discrepancy
might support a more complicated scenario than that envisioned in our hypothesis, or it might
indicate limited statistical resolution given only three competition assays performed for each
combination of clone and treatment. To gain better resolution of changes in fitness over this
critical period, we competed the Cit– clones from 33,000 and 34,000-generations against one
another, using a neutral genetic marker to distinguish them (see Methods and Table S1).
Consistent with our predictions, the later C4-dicarboxylate-consuming clone had a fitness of 1.14
± 0.04 (mean ± 95% confidence interval, n = 10) relative to the earlier clone that could not use
those substrates, when they competed in the presence of Cit+ cells. Moreover, in the absence of
the Cit+ cells, the 34,000-generation Cit– clone had a fitness of only 0.94 ± 0.02 relative to the
33,000-generation Cit– clone, providing further support for the hypothesis that adaptation of the
17
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Cit– cells to growth on C4-dicarboxylates imposed a tradeoff with respect to their performance in
their original glucose-only niche.
Genomic analysis
We compared the sequenced genomes of seven Cit– clones that differed in their capacity for
growth on C4-dicarboxylates, generating a list of 19 distinguishing mutations that might underlie
that phenotype (Table S2). We performed Sanger sequencing to determine the presence or
absence of many of these mutations in six additional Cit– clones: CZB193, CZB194, and
CZB195, which did not grow on C4-dicarboxylates; and ZDB86, ZDB88, and ZDB92, which did
grow on those substrates. These additional data narrowed the list of candidates to nine mutations
(Table S2). Of all these candidates, the 5-bp deletion in the dcuS gene is the only one that has
clear relevance to the C4-dicarboxylate growth phenotype. The DcuS protein is a C4dicarboxylate sensor that induces expression of the dctA gene, which in turn encodes DctA, a
dicarboxylic-acid transporter protein (Janausch et al. 2004). The founding strain of the LTEE had
a 5-bp insertion in dcuS (Jeong et al. 2009), which eliminated the DcuS function and thereby
prevented expression of the dctA gene and its encoded dicarboxylic-acid transporter (Yoon et al.
2012; Quandt et al. 2014).
Genetic manipulation
We tested whether the deletion in dcuS was, in fact, a gain-of-function mutation that conferred
on the Cit– lineage the ability to grow on the C4-dicarboxylates that were secreted by the Cit+
lineage. To do so, we constructed ZDB1052, an isogenic derivative of the ancestor REL606
except with the dcuS allele from a 34,000-generation Cit– clone called ZDB86. We then
18
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measured the growth over 48 h of REL606, ZDB86, and ZDB1052 on succinate, fumarate, and
malate.
ZDB1052 had improved growth on succinate, fumarate, and malate compared to REL606
(Fig. 7A-C). The densities achieved by ZDB1052 on these substrates after 48 h were comparable
to those reached by ZDB86, the earliest Cit– clone in our study to exhibit the C4-dicarboxylate
growth phenotype and the source of the evolved dcuS allele. However, ZDB1052 grew more
slowly on the C4-dicarboxylates than did ZDB86. ZDB1052 also grew more slowly on glucose,
and reached a slightly lower final density on that substrate, than did REL606, its counterpart with
the ancestral dcuS allele (Fig. 7D). This result provides direct evidence that adaptation to growth
on the C4-dicarboxylates caused a tradeoff in terms of growth on glucose, the sole carbon source
that was available to the ancestral bacteria. ZDB86 does not show any indication of that tradeoff
(Fig. 7A-D), but that is not surprising because it contains many mutations that were beneficial
for growth on glucose, which had fixed before citrate utilization evolved and C4-dicarboxylates
appeared.
The ancestral strain, REL606, did not grow appreciably on C4-dicarboxylates during the
24-h interval between transfers of the LTEE, but it did show slight growth between 36 and 48 h
on two of these compounds (Fig. 7A-C). This pattern suggests that growth on C4-dicarboxylates
has a long lag and is slow, but not absent, in the ancestral genotype. Alternatively, this growth
could have resulted from a mutant with the ability to grow on C4-dicarboxylates. We did not try
to distinguish these alternatives because the purpose of collecting these data was to test whether
the evolved dcuS allele led to improved growth on C4-dicarboxylates, which it clearly did.
Discussion
19
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The ability to grow aerobically on citrate evolved in only one of the twelve LTEE populations
(Blount et al. 2008), even after 60,000 generations and billions of spontaneous mutations in each
population (Lenski 2004). This evolutionary innovation provided access to a previously
unavailable resource, and it caused dramatic ecological changes in the flask-based ecosystem
where it arose. Owing to the abundant citrate in the medium, the total population experienced a
several-fold increase in density when the Cit+ cells became dominant in the population.
However, the Cit+ lineage did not eliminate all of the non-citrate consumers from the population.
Instead, two ecotypes, one Cit+ and one Cit–, coexisted for thousands of generations in a
frequency-dependent manner. We sought to understand the ecological dimensions of this
prolonged coexistence. Based on several lines of evidence, we discovered that the Cit– lineage
was able to persist, at least in part, by evolving a cross-feeding relationship with the Cit+ lineage,
one in which three C4-dicarboxylates were the secreted metabolites.
We ruled out the possibility that the Cit– lineage coexisted with the Cit+ lineage by
improvements in their growth on the exogenously supplied glucose. Although Cit– cells initially
had an advantage over Cit+ cells in competition for glucose (Blount et al. 2008), improved
growth on glucose did not sustain the Cit– lineage. In fact, the fitness of the Cit– lineage in the
standard glucose-limited medium, when measured in the absence of a Cit+ population, actually
declined over evolutionary time (Fig. 6).
However, the fitness of clones from the Cit– clade increased over evolutionary time when
they competed against their predecessor in the presence of Cit+ cells (Fig. 6). This finding
supports our hypothesis that the Cit– lineage adapted to changes in the environment caused by
the activity of Cit+ cells. In particular, Cit+ cells secreted the C4-dicarboxylates succinate,
20
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fumarate, and malate into the culture medium, whereas Cit– cells (including predecessors from
the clade that generated the Cit+ lineage) did not (Fig. 1). The earliest Cit+ clone that we tested
showed only weak growth on citrate (Blount et al. 2012), but it, too, released C4-dicarboxylates
into the medium. However, it did not fully draw them back down during the course of the 24-h
transfer cycle, whereas Cit+ clones from later generations both released these C4-dicarboxylates
and then drew them down below detectable levels. Although the observed concentrations of the
endogenously produced succinate, fumarate, and malate were low relative to the exogenously
added concentrations of glucose and citrate, the total amounts of C4-dicarboxylate secreted and
available to support growth would probably be much higher if they could be integrated over the
course of an entire cycle. In fact, the citrate transporter protein CitT transports one molecule out
of the cell for each molecule that it transports into the cell (Pos et al. 1998). While some of these
antiporter events may have involved the exchange of one citrate molecule for another, the Cit+
populations (except in their earliest form) eventually consumed all of the citrate that was
available each day (Fig. 2). It follows logically, then, that the total quantity of C4-dicarboxylate
molecules released into the medium was equal to the number of citrate molecules consumed,
implying substantial opportunity for growth on C4-dicarboxylates and, consequently, strong
selection to improve growth on those resources. C4-dicarboxylate concentrations were generally
highest from about 4 to 6 h after inoculation, which coincided with the period when citrate was
being drawn down from the medium (Fig. 2), corroborating the biochemical evidence (Pos et al.
1998) that the release of C4-dicarboxylates is simultaneous to citrate uptake.
In addition to showing that the Cit+ population released C4-dicarboxylates into the
medium, and that the Cit– population was becoming more fit in the presence (but not the
absence) of the Cit+ population, we also tested explicitly whether the Cit– lineage evolved to
21
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exploit those C4-dicarboxylates. Indeed, we found that C4-dicarboxylate consumption by the Cit–
lineage evolved in close temporal proximity to the rise of the Cit+ population to numerical
dominance between 33,000 and 33,500 generations. Clones isolated from the Cit– lineage
through 33,000 generations showed no measurable growth on succinate, fumarate, or malate, but
clones from 34,000 generations and later grew well on all three C4-dicarboxylates (Fig. 3).
Moreover, the time for populations to reach stationary phase, which is a function of both the
length of the lag phase and the subsequent growth rate, declined in later generations, indicating
continued adaptation of the Cit– population to growth on C4-dicarboxylates. This improvement
was not a generalized improvement in fitness because the growth of the Cit– clones on both
glucose and acetate declined over this period.
The Cit+ lineage also evolved improved growth on C4-dicarboxylates, although that
improvement was somewhat more sporadic than the improvements seen in the Cit– lineage (Fig.
4). The earliest clone able to grow on citrate, from generation 32,000, did not show any
detectable growth on C4-dicarboxylates, indicating that improved growth on these compounds
was not simply a pleiotropic effect of the ability to consume citrate. The greater variability of
growth on C4-dicarboxylates in the Cit+ lineage suggests that selection to use them was either
weaker or less effective in this lineage than in the Cit– lineage. That variability may reflect the
fact that Cit+ cells can also metabolize citrate, a more abundant and energetically valuable
resource than any of the C4-dicarboxylates, so that mutations that improve growth on citrate
might be favored even if they reduce growth on one or more of these byproducts.
In both the Cit+ and Cit– lineages, the mechanism underlying the improved growth on C4dicarboxylates appears to be increased expression of the DctA transporter protein. DctA is a
proton motive force-driven transporter that E. coli requires for transport of C4-dicarboxylates
22
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during aerobic metabolism (Davies et al. 1999). The ancestral strain of the LTEE had a 5-bp
frameshift mutation in the dcuS gene, which encodes a regulator that promotes expression of
DctA (Jeong et al. 2009; Quandt et al. 2014). In the Cit+ lineage, previous work showed that a
mutation in the promoter region of the dctA gene caused increased DctA expression and restored
the ability to grow on C4-dicarboxylates (Quandt et al. 2014). Similarly, in the Cit– lineage, we
have showed that a 5-bp deletion in dcuS that restored its reading frame enabled growth on C4dicarbolyates (Fig. 7).
The consumption by Cit– cells of the C4-dicarboxylates released by Cit+ cells is an
example of cross-feeding, a phenomenon that has been observed in several previous microbial
experiments (Rosenzweig et al. 1994; Treves et al. 1998). Cross-feeding typically occurs when
one organism evolves to grow faster by partially degrading a primary resource, leaving a
secondary resource for another organism to consume. In this canonical scenario, incomplete
degradation of the primary resource is adaptive because it leads to faster growth on the primary
resource (Doebeli 2002; Pfeiffer and Bonhoeffer 2004). However, the evolution of cross-feeding
between the Cit+ and Cit– lineages follows a somewhat different pattern. The release of C4dicarboxylates by the Cit+ cells does not itself appear to be beneficial to them. Indeed, while the
Cit– lineage evolved increased growth on the C4-dicarboxylates, so too did the Cit+ lineage.
Thus, unlike the typical cross-feeding relationship, the two ecotypes have evolved increased, not
decreased, overlap in their resource usage. Instead of being an adaptive trait, cross-feeding in this
case appears to reflect evolutionary ‘bricolage’ (Jacob 1977), whereby evolution tinkers with
existing components to solve new problems.
The Cit+ lineage studied here evolved the ability to consume citrate via multiple
mutations including, in particular, a complex rearrangement that allowed aerobic expression of
23
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an existing CitT transporter (Blount et al. 2012). Most E. coli cells can express the CitT protein
only under anaerobic conditions, where cells ferment citrate into succinate (but only when they
also have another growth substrate in addition to citrate). Under anaerobic conditions, the use of
a transporter that exchanges citrate for succinate has a clear benefit because succinate is an end
product of fermentation. However, in the Cit+ lineage, the CitT transporter is expressed under
carbon-limited, aerobic conditions where succinate and other C4-dicarboxylates provide a useful
source of energy and carbon. Many other bacterial species use proton or sodium gradients for
citrate transport (Boorsma et al. 1996; Bott 1997). Furthermore, in the only other reported case
where mutations gave rise to aerobic consumption of citrate by E. coli (Hall 1982), the citrate
importation appears to have been powered by the proton motive force (Reynolds and Silver
1983), although the genetic and mechanistic bases are unknown in that case. If the Cit+ ecotype
in the LTEE had instead evolved to use a proton or sodium gradient for citrate uptake, then it
would not have exported C4-dicarboxylates. A hypothetical Cit+ mutant that employed a proton
or sodium gradient would probably be favored over the evolved Cit+ bacteria that release
valuable molecules that are often lost to competitors. Instead, as a consequence of evolutionary
tinkering, or bricolage, the Cit+ lineage first evolved to express the previously unexpressed CitT
antiporter under aerobic conditions (Blount et al. 2012) and then to reabsorb the exported C4dicarboxylates via the proton motive force by using the previously unexpressed DctA transporter
(Quandt et al. 2014). While this solution seems not to have been the most elegant or efficient, it
nonetheless proved highly successful for the Cit+ clade. At the same time, this solution provided
an opportunity for the Cit– lineage to expand its niche as well, transitioning from being a glucose
specialist to also consuming some of the C4-dicarboxylates released by the Cit+ cells.
24
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Acknowledgments
We thank Justin Meyer for assistance with λ phage; Neerja Hajela for laboratory assistance;
Mike Wiser, Alita Burmeister, and Rohan Maddamsetti for helpful comments on this paper; and
the technical staff at the MSU Mass Spectrometry Core Facility for assistance with GCMS. This
research was supported, in part, by EPA STAR and MSU Distinguished Graduate Student
Fellowships to C.B.T., an NSF grant (DEB-1019989) to R.E.L., a grant from the John Templeton
Foundation to R.E.L. and Z.D.B., and the BEACON Center for the Study of Evolution in Action
(NSF Cooperative Agreement DBI-0934).
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Malate concentration (mg/L)
Fumarate concentration (mg/L)
Succinate concentration (mg/L)
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2.5
A.
2.0
1.5
1.0
0.5
0.0
Cit- (40,000 generations)
0 Generations
30,000 generations
32,000 generations
34,000 generations
36,000 generations
38,000 generations
40,000 generations
42,000 generations
44,000 generations
-0.5
0
4
8
12
16
20
24
1.4
B.
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
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20
24
12
C.
10
8
6
4
2
0
-2
0
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8
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24
Time (hours)
Figure 1. Concentrations of succinate (A), malate (B), and fumarate (C) in filtered medium
sampled over a 24-h transfer cycle. Red points and lines show clones with the Cit– phenotype,
purple shows a weakly Cit+ clone, and blue shows strongly Cit+ clones.
30
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Citrate concentration (mg/L)
400
300
200
100
0
0
5
10
15
20
25
Time (h)
Figure 2. Citrate concentration in filtered medium sampled over a 24-h transfer cycle. Red
points and lines show a Cit– clone, purple shows a weakly Cit+ clone, and blue shows two
strongly Cit+ clones. Error bars are 95% confidence intervals.
31
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OD420 at 24 h
Succinate
0.03
0.03
0.03
0.02
0.02
0.02
0.01
0.01
0.01
0.00
0.00
0.00
A.
-0.01
C.
B.
-0.01
30 32 34 36 38 40 42 44
Time to stationary phase (h)
Malate
Fumarate
-0.01
30 32 34 36 38 40 42 44
30 32 34 36 38 40 42 44
25
25
25
20
20
20
15
15
15
10
10
10
5
5
5
D.
0
0
30 32 34 36 38 40 42 44
F.
E.
0
30 32 34 36 38 40 42 44
30 32 34 36 38 40 42 44
Generations (thousands)
Figure 3. Final OD at 420 nm (A-C) and time to stationary phase (D-F) of clones isolated from
the Cit– clade at the various generations indicated grown in DM medium supplemented with 30.5
mg/L succinate, 39.5 mg/L fumarate, or 45.7 mg/L malate. Error bars are 95% confidence
intervals.
32
bioRxiv preprint first posted online Jun. 17, 2015; doi: http://dx.doi.org/10.1101/020958. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
OD420 at 24 h
Succinate
Malate
Fumarate
0.05
0.05
0.04
0.04
0.03
0.02
0.03
0.03
0.02
0.02
0.01
0.01
0.01
0.00
0.00
0.00
A.
C.
B.
-0.01
-0.01
Time to stationary phase (h)
30 32 34 36 38 40 42 44
30 32 34 36 38 40 42 44
30 32 34 36 38 40 42 44
25
25
25
20
20
15
15
15
10
10
10
5
20
5
5
D.
0
0
30 32 34 36 38 40 42 44
F.
E.
0
30 32 34 36 38 40 42 44
30 32 34 36 38 40 42 44
Generations (thousands)
Figure 4. Final OD at 420 nm (A-C) and time to stationary phase (D-F) of clones isolated from
the Cit+ clade at the various generations indicated grown in M9 medium supplemented with 30.5
mg/L succinate, 39.5 mg/L fumarate, or 45.7 mg/L malate. Open and filled circles show clones
with Cit– and Cit+ phenotypes. Error bars are 95% confidence intervals.
33
bioRxiv preprint first posted online Jun. 17, 2015; doi: http://dx.doi.org/10.1101/020958. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Cit0.04
A.
OD420 at 24 h
0.03
0.02
0.01
0.00
-0.01
Time to stationary phase (h)
30 32 34 36 38 40 42 44
B.
30
25
20
15
10
5
0
30 32 34 36 38 40 42 44
Generations (thousands)
Figure 5. Final OD at 420 nm (A) and time to stationary phase (B) of clones from the Cit– clade
growing in DM medium supplemented with 30.5 mg/L acetate. Error bars are 95% confidence
intervals.
34
bioRxiv preprint first posted online Jun. 17, 2015; doi: http://dx.doi.org/10.1101/020958. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
2.0
1.8
Fitness
1.6
1.4
1.2
1.0
0.8
0.6
0.4
30
32
34
36
38
40
42
44
Generations (Thousands)
Figure 6. Fitness of clones isolated from the Cit– clade at the various generations indicated
relative to a 30,000-generation clone from the Cit– clade. Filled and open symbols indicate
competitions between the Cit– clones that were performed in the presence and absence,
respectively, of a 40,000-generation Cit+ clone. Error bars are 95% confidence intervals.
35
bioRxiv preprint first posted online Jun. 17, 2015; doi: http://dx.doi.org/10.1101/020958. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
B. Fumarate
OD420
A. Succinate
0.04
0.04
0.03
0.03
0.02
0.02
0.01
0.01
0
0
0
12
24
36
0
48
12
36
48
36
48
D. Glucose
C. Malate
OD420
24
0.04
0.04
0.03
0.03
0.02
0.02
0.01
0.01
0
0
0
12
24
36
48
0
12
24
Time (h)
Time (h)
Figure 7. Growth curves showing OD at 420 nm of the ancestral strain (REL606, black squares),
a 34,000-generation Cit– clone (ZDB86, blue triangles), and a modified ancestral strain with the
evolved dcuS allele (ZDB1052, red circles) grown in DM medium supplemented with (A) 30.5
mg/L succinate, (B) 39.5 mg/L fumarate, (C) 45.7 mg/L malate, or (D) 25 mg/L glucose.
36